After being contraindicated, physical activity take an increasingly important place in the management of multiple sclerosis (MS). The training at mild to moderate intensity is widely documented, contrary to the high intensity training. This work has focused on the effects of high intensity interval training combined with resistance training during 12 weeks in 26 MS patients. Our results show that peak oxygen consumption, maximum tolerated power, isokinetic muscle strength in both quadriceps and hamstrings and quality of life are improved. Women show more important and more numerous improvements than men, however, the disability level does not seem to limit improvements. Our work has demonstrated that high intensity interval training combined with resistance training is well tolerated and allows physical capacities and quality of life improvements.

Ribosomes are ribo-nucleoprotein complexes that read mRNA to synthesize proteins. This is important to consider in the case of cancer cells, which show a global increase in protein synthesis to support their hyper-proliferative behaviour. Inhibitors of eukaryotic protein synthesis have been shown to have significant therapeutic potential to treat a wide range of human cancers. Recently we published a multidisciplinary study in which we unravel the mechanism of action of chlorolissoclimide (CL), a compound sharing chemical similarity to cycloheximide (CHX), but showing promising lower cytotoxicity. We were interested in the new types of interaction of CL with the ribosome and therefore we solved a new crystal structure of the S.cerevisiae 80S in complex with another lissoclimide compound at 3.1 Å resolution. The coupling of high-resolution structures with computationally-driven drug design and FRET analysis will further guide the design of more selective and less cytotoxic inhibitors.

One of the main limits in liver surgery is the risk of liver failure (LF) after hepatectomy (Hx) or graft dysfunction (EAD) after liver transplantation (LT). Few studies have evaluated global liver metabolism, probably due to the lack of clinically relevant techniques. HR-MAS-NMR metabolomics may fulfill this lack and the goal of this work was to evaluate its capacity to predict early outcomes after hepatectomy and LT. In LT (n=42), metabolic profile predicted EAD and lactate and phosphocholine were potent biomarkers providing means for metabolic matching. In liver biopsies harvested at the end of major Hx (n=45), metabolic profile predicted PHLF. The profile at risk of LF differed from that of decompensated cirrhosis but correlated to that of proliferative multicellular systems. A preliminary study showed that the metabolic profile predicted the risk of liver metastases recurrence at 1 year. This work underlines the potential value of HR-MAS-NMR metabolomics in the prediction of short-term outcomes in liver surgery. It provides clues to be further investigated for future evaluation of metabolic intervention in the field of liver surgery.

Plants are sessile organisms that are not able to escape from difficult environmental conditions and therefore have to adapt to multiple abiotic and biotic stress to survive. Cutin is a part of the cuticle which plays a major role as a barrier for the plant. It’s a lipid polymer composed mainly by hydroxylated and epoxidized C16 and C18 fatty acids linked together by ester links involving the carboxyl and ω-hydroxyl functions of those fatty acids. Cutin plays also a role as a reservoir of molecules with fundamental physiological properties. With biochemical and genetic approaches, we characterized AtEH1, an epoxide hydrolase responsible for the formation of diols incorporated in Arabidopsis thaliana cutin. These diols are described as being involved in plant-pathogen interactions. We also showed that these compounds as well as others fatty acids derivatives are perceived by plants. We have also identified and characterized CYP77B1, an epoxidase that has a potential role in the formation of polyhydroxylated fatty acids incorporated in cutin.

Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II (RNA Pol II) transcription with about 10% of genes in S. cerevisiae dependent on SAGA. The remainder 90% of the genome would be regulated by TFIID. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We observed that SAGA maps to regulatory elements of most genes, irrespective of previous designations of SAGA- or TFIID-dominated genes. Additionally, disruption of either SAGA or TFIID through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly-synthesized RNA or RNA Pol II chromatin immunoprecipitation. We also found that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that both SAGA and TFIID act as general cofactors required for essentially all RNA Pol II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.

Backcross-based introgression has been used in French breeding programmes to transfer resistance factors from Muscadinia rotundifolia into cultivated grapevine Vitis vinifera while eliminating the unwanted cultural traits and off-flavours. However, crosses between the two species have a low success rate. In this context, it is essential to identify (1) the genomic features that impede crosses between the two species, (2) the traits specific to M. rotundifolia that have to be eliminated during crosses. To this end, morphology, phenology and metabolites of leaves and berries were compared in accessions of both species to identify traits specific to M. rotundifolia. Genotyping-by-sequencing was used to establish high-density genetic linkage maps of three mapping populations generated by pseudo-backcrosses and to determine the origin of each segment of the chromosomes. These maps were used to study variation of recombination rate along the chromosomes and to establish the genetic determinism of the traits specific to M. rotundifolia.

7.
Olivares, Elodie.
Évaluation de l'impact des antibiotiques sur la formation de biofilms par P. aeruginosa : place de l'Antibiofilmogramme® : Evaluation of the impact of antimicrobials on the biofilm formation by P. aeruginosa : place of the Antibiofilmogram®.

Cystic fibrosis (CF) patients are predisposed to chronic colonisation of the upper airways by P. aeruginosa. This opportunist pathogen is characterized by its ability to adhere to a surface and to form a protective biofilm, which is highly tolerant to antimicrobials. In routine, antibiograms are realised on planktonic bacterial cultures. The efficacy of the corresponding antimicrobial therapies appears low for the eradication of bacterial biofilms. The realisation of Antibiofilmograms® on CF clinical isolates (a new tool investigating the susceptibility of sessile bacteria to antibiotics) highlighted phenomena of biofilm formation inhibition and induction. More precisely, aminoglycosides are able to delay the bacterial adherence. Conversely, the β-lactam family shows the ability to stimulate the early adhesion of microorganisms. These different effects of antimicrobials on the bacterial behaviour are confirmed with more conventional in vitro methods (Crystal Violet, enzymatic treatment with DNase I) and a cell model (static co-culture of eukaryotic cells and bacteria). The clinical relevance of the Antibiofilmogram® is reinforced by its ability to detect the initiation of the early bacterial adhesion, to select inhibitor molecules and to avoid the inducer ones. Associated to traditional antibiograms, its application should be pertinent to optimise the CF therapies for the…

Community life gives many advantages, but also disadvantages with which animal must accommodate. In order to maintain social cohesion, group members must therefore make compromise and resolve quickly conflicts of interest. Understanding how individuals reach common and optimal decisions helps to learn more about the social organization, movement patterns and habitat use of the species. In return, this information is essential to the development of new management strategies. In this context, I was interested in group life and collective movements of the European bison, species which experiencing its great come back on its continent of origin. My works have shown that (1) matrilineal ties were at the core of the species’ social life, (2) the European bison, contrary to popular opinion, was far from a strictly forest animal and (3) group movements were preferentially lead by the oldest dominant females which had a calf. Taking into account these results could be greatly improve the reintroduction and management of bison populations and, thereby, allow a better cohabitation with humans.

Cerebellum is a brain structure involved in motor regulation and motor learning. In the cerebellar cortex, sensorimotor information is transmitted by granule cells. During my PhD, I demonstrated that the properties of individual granule cell synaptic connections are highly heterogeneous. From one synapse to another, I observed ultrastructural, molecular and functional variability at unitary contacts. More precisely, I assessed the properties of short term plasticity at individual synapses during high frequency trains of stimulation :1) Short term plasticities are highly heterogeneous from one synapse to another and can be classified in sub-categories.2) Some categories of short-term plasticity profiles relie on the expression of molecules such as Synapsin2.3) The response of post-synaptic neuron to high-frequency inputs is dependent on the nature of the activated synaptic contact.

Patients suffering from schizophrenia display clinical symptoms as well as cognitive deficits. Recently, it has been suggested that these patients display, among other things, reading deficits. This doctoral thesis aims to evaluate the abilities of visual word recognition in patients suffering from schizophrenia, using both behavioral and electrophysiological (recording of event related potentials) approaches. The results indicated that the specialization of the visual word form area for processing of written words is preserved for patients. In addition, cognitive processes involved in orthographic processing of letter strings were preserved. By contrast, cognitive processes involved in phonological processing were altered for patients suffering from schizophrenia.

Cancer gene therapy requires the use of an effective suicide gene and the specific targeting of cancer cells. In my PhD work, I have first characterized a new potential suicide gene derived from human deoxycytidine kinase (dCK): M36. Compared to dCK, M36 improves sensitization of certain cancer cells to treatment with chemotherapeutic compounds as gemcitabine and AraC. These results are particularly encouraging for the elimination of cancer cells resistant to the treatment because of a defect with dCK. In a second part, I have worked at the proof of concept that a modified HIV envelope can allow specific targeting of cancer cells by lentiviral vectors. During this work, I have generated a CD4i envelope with a strongly diminished natural tropism and that carries a motif known to bind the model cell surface cancer marker HER2. This envelope constitutes a good starting material to be improved by evolution in cell culture to obtain specific targeting of HER2+ cells.

The purpose of this project was to gain more knowledge about cognitive control mechanisms underlying deteriorations and fluctuation of sustained attention in schizophrenia and healthy participants. To that end, we combined the use of behavioral, electrophysiological (event-related potentials and functional connectivity) and subjective measures. Our results revealed spared sustained attention in schizophrenia and a distinct patterns of sustained attention changes in schizophrenia. Deteriorations are underlined by a decrease of reactive mode of cognitive control in patients and by a decrease of proactive mode in controls. Our results also highlighted slightly distinct patterns of precursors of lapses in sustained attention in schizophrenia according to the attentional state. Sustained attention changes are associated with resource depletion in patients, whereas in healthy participants, according to attentional state, they could also be caused by disengagement of cognitive control.

Metastasis can be considered as the end product of a multistep bio-mechano-chemical process where cancer cells disseminate to anatomically distant organs and home and establish themselves in a new tissue microenvironment. Although metastasis is the leading cause of cancer-related death, the main cellular mechanisms enabling this process remain to be elucidated. Importantly, the scientific community lacks adapted imaging technologies to accurately dissect, at the highest resolution possible, tumor cell behavior in vivo. Therefore, the main goal of my PhD thesis was to develop an intravital and non-invasive imaging approach to track tumor progression in the living mouse. This approach was included in the development of an intravital Correlative Light and Electron Microscopy protocol allowing to track tumor cells at different scales in their natural environment. It was used to study single invasive tumor cells in the mouse ear and brain and to describe the details of cell protrusions and cell-matrix interactions during invasion and intravasation of cancer cells.

Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in the HECT domain of the E3 ubiquitin ligase NEDD4L lead to PNH associated with toes syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed a sensitivity of PNH-associated mutants to proteasome degradation. Moreover, in utero electroporation approach showed that PNH-related mutants and excess of wild type (WT) NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin based experiments, revealed differential deregulation of pathways involved. Excess of WT NEDD4L leads to a disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with a deregulation of mTORC1 and AKT activities. Altogether, these data provide insights to better understand the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.

The synthesis of messenger RNA is a highly regulated process. During transcription initiation, a large number of proteins are recruited to gene promoter, including the RNA polymerase II, general transcription factors, co-activators, chromatin remodellers and the Mediator complex. Some DNA repair factors from the NER pathway are also recruited. Using cells derived from patients bearing mutations in either MED12 gene or XPC gene, we studied the roles of such proteins in transcription. MED12 patients are mostly characterised by intellectual disability and developmental delay. We showed that MED12 is implicated in the transcription regulation of immediate early genes like JUN, known for its role in neurological development and neuronal plasticity. JUN expression is markedly altered by MED12 mutations. We also showed that the position of the mutation influences this alteration, bringing possible explanation for inter-patients symptom variability. Meanwhile, XPC patients are mostly characterized by photosensitivity. We showed that XPC protein, which engages one of the NER pathways, is implicated in chromatin post-translational modification. Together with E2F1, it helps the…

The mevalonate (MVA) pathway is an essential metabolic pathway that leads to the production of molecules important in several physiological processes and play pivotal roles in the brain. An imbalance of this pathway in CNS is accompanied by the onset of several neuropathological descriptions. Despite these observations, the physiological importance of this metabolic process in the brain has remained unclear. My aim was to study the presence and the regulation of the proteins involved in the MVA pathway in different rat brain areas in a sex- and age-dependent manner, to analyze the impact of the key enzymes on neuronal development and on rat behavior, and to explore whether the MVA pathway is affected in a neurodevelopmental disease such as autism. My results demonstrate that this metabolic process is expressed and modulated in a highly region-dependent manner and that age and sex induce physiological differences. Notably, it impact on behavior and neuronal development suggesting that this pathway may be considered as potential molecular target when designing novel therapeutic approaches for the treatment of these pathologies.

Chitotriosidase (CHIT1) is a human chitinase belonging to the glycosyl hydrolase family 18 (GH18), a highly conserved enzyme family. GH18 enzymes hydrolyze chitin, a N-acetyl glucosamine polymer. CHIT1 is characterized by many enzymatic features that are conserved in GH18 and not completely understood. To increase our knowledge on the catalytic mechanism in CHIT1 and GH18 family, I improved the X-ray resolution crystal structure of CHIT1 catalytic domain in apo and pseudo apo forms as well as in complex with a synthetic substrate to a resolution range between 0.95Å and at 1.10Å. My results allow me to suggest a new mechanism for chito-oligosaccharide chains hydrolysis. Moreover, thanks to a new a crystallogenesis strategy, I obtained the first crystal structure of full length CHIT1 at 1.95Å resolution. My study presents many structural and mechanistic aspects of CHIT1 which gives new insights onto its mode of action and shed light into the conserved enzymatic features in GH18 chitinase family.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins catalyzed by poly(ADPribose) polymerases (PARPs). PARP3 was identified as a novel actor of the double-strand break (DSBs) repair pathway. We evaluated the contribution of PARP3 in these repair pathways(HR, C-NHEJ ou A-EJ). Our results defined PARP3 as a modulator of the single strand DNA resection process which plays a role in driving the repair pathway choice. We showed that PARP3 enhances the recruitement of the Ku70/Ku80 complexe to damaged sites and modulates the BRCA1/53BP1 balance. These two events prevent the DNA end resection step initiating HR and A-EJ and drives the repair towards the C-NHEJ. By chromatin immunoprecipitation, we studied the consequences of the absence of PARP3 on histone modifications, known to modulate the decision of the DSBs repair pathways. Our current results didn’t allow us to establish a link between PARP3 and histone modifications in response to DSBs. However, in absence of DNA damage and PARP3, we observed an accumulation of H3K36me2, a histone mark known to regulate transcriptionally active genes. In a second project, we studied the impact of the absence of PARP3 on cell viability and tumor progression in breast cancer cell lines mutated in BRCA1. By in vitro and…

Background: Older adults constitute one of the most rapidly growing population groups. Consequently, avoiding an inactive lifestyle and encouraging regular physical activity remains one of the main measures that should be promoted for older adults. The main objective of our study was to determine the effects of a new lightweight protocol including recovery bouts called: “The Intermittent Aerobic Training Program with Recovery bouts” (IATP-R - PEP’C-R in French) for seniors over 70 on maximal cardiorespiratory and endurance parameters. The secondary objectives were to determine the effects of IATP-R on vascular function and cognitive performances. Results: Our results showed that this new “IATP-R” training protocol permits to achieve a significant improvement in both endurance parameters and maximal cardio-respiratory parameters. In addition, the IATP-R permits to improve the vascular function and cognitive performances. Conclusion: The results of this study could be useful to implement this new IATP-R protocol for healthy older adults over 70. This would also be within the framework of prescribing training programs.

Identification of risk factors for venous thromboembolism (VTE) is a major concern for the prevention of the disease and its recurrence. We have shown in humans that the cumulative cardiovascular risk factors (CVFR) for atherosclerosis were associated with unprovoked VTE, its severity and the risk of recurrence. Aging, CVRF and VTE risk factor, induced an endothelial dysfunction in the rat femoral vein involving cyclooxygenases (COX) associated with potential pro-thrombogenic prostanoids generation. In the young obese ZSF1 rat, cumulative CVRF induced early venous endothelial dysfunction related to COX-1 and 2. Omega-3 treatment in aging rats modulates arachidonic acid pathway leading to COX-2-mediated formation of less deleterious prostanoids, associated with a decreased thrombogenic risk, making omega-3 a potential adjuvant treatment for VTE.

Habitats and landscape fragmentation, caused by linear land transports infrastructures, is one of the major cause for the current loss of biodiversity. Among those infrastructures, road is a major cause of fragmentation, especially as it possess specific traffic-linked effects, which induces wildlife-vehicles collisions and landscape pollution. In order to decrease those negative effects, mitigation measures are taken, among which wildlife crossings, enabling wildlife to cross the road. Road also creates new potential habitats for small wildlife species in anthropogenic and fragmented landscapes. In this essay are shown (1) the potential as habitat of different road-linked elements; (2) the possibility to anticipate wildlife-vehicles collisions in order to improve the position of mitigation measures; (3) the importance of methodology in the evaluation of wildlife crossings effectiveness; and (4) the possibility to improve existing wildlife crossings. Those results will allow improving landscape defragmentation strategies.

The energetic cost of foraging activities in King Penguin (Aptenodytes patagonicus) consists to reach favourable areas, realizes depth diving to attempt fish patch and resting in high latitude cold water. Several studies have shown that resting in cold water could be represent a more expensive cost than realized depth diving. Indeed, this paradox is probably linked with contrasting thermoregulation processes. During daylight, a general hypothermia occurs and is believed to reduce energy expenditure. At sunrise occurs a re-warming to normothermia, contributing to increase heat-loss during the night. We hypothesise an energetic conflict between thermoregulation and digestive processes. During daylight, the organism may be unable to assimilate the end product of prey digestion (free fatty acids) inside the peripheral subcutaneous adipose tissues (SAT), because skin is no more blood perfused. During the night, re-warming and re-connecting to blood circulation peripheral tissues could be inevitable to end the assimilation of FFA inside the SAT. In a first step, we have reproduced the conditions of a resting night at sea and events of rewarming skin temperature, using a sea water tank in which king penguins equipped with internal temperature loggers were maintained several days. In a second step, we have tested a generalisation of our hypothesis studying body temperature variations on penguins fast and feed. Finally, we have measured the cost to maintain normothermia in cold water with respirometry measures and investigated peripheral vasodilation with body temperature variations and infrared thermography.

An endogenous spinal cholinergic tone modulating nociceptive (pain­like) behaviors has been demonstrated in rodents and humans. One potential source of this acetylcholine is the spinal Dorsal Horn (DH) cholinergic interneurons. Our objectives were to: (1) characterize the spinal cholinergic tone establishing mechanical nociceptive thresholds and (2) to elucidate the role of DH cholinergic neurons in the modulation of sensory information of naïve and neuropathic animals. We have confirmed the presence of a cholinergic tone modulating mechanical thresholds and demonstrated that it is still present, although altered, after neuropathy. The DH cholinergic interneurons receive excitatory inputs from distant spinal segments and generally receive lower inhibitory inputs. In addition, they are indirectly connected by a subset of nociceptive primary afferents expressing TRPV1, demonstrating their involvement in nociceptive processing. In neuropathic spinal circuits, the inputs to LIII/IV neurons appears to be unaffected after injury. Better understanding the spinal cholinergic system can pave way to alternative pain therapy.

The use of glucocorticoids (GC) to treat inflammatory diseases or androgen antagonists for prostate cancer is limited by the occurrence of side effects such as muscle atrophy. As the underlying mechanisms were unclear, we characterized the effects of GC and androgens on muscle mass and function. Our results demonstrate that myofiber GC receptor negatively controls muscle mass by distinct actions under physiological and pharmacological levels of GC. Moreover, our data identified many genes and networks controlled by GC in myofibers. We also showed that androgens promote the gain in muscle performance during postnatal development via the improvement of specific maximal force and power. Thus, this study allowed to clarify the molecular and cellular mechanisms regulating muscle homeostasis, and paves the way to identify new therapeutic targets.

Inflammatory myopathies are rare autoimmune diseases whose common denominator is muscle weakness and inflammation. Their origin is not known and conventional treatments are partially effective. Using an epidemiological approach, we have shown that the study of incidence and prevalence is an useful tool for identifying determinants of inflammatory myopathies. However, better identification and classification of patients is mandatory to refine the epidemiology of inflammatory myopathies. Using a translational approach, we have shown that, compared with other inflammatory myopathies, perifascicular mitochondrial dysfunctions are a characteristic of dermatomyositis, which play a role in exercise intolerance and in the maintenance of inflammation. These results open up new avenues to better understand and treat inflammatory myopathies.

C35 Bacteriohopanoids represent the majority of hopanoids produced by bacteria. They bear an additional C5 side chain linked by a carbon/carbon bond to the isopropyl group of the hopane skeleton. The C5 side chains present an impressive structural diversity and carry taxonomic and physiological information. The most common C35 bacteriohopanoids are bacteriohopanetetrol (BHT) and aminobacteriohopanetriol. Moreover, these two compounds are proposed as the parents of most complex bacteriohopanoids. Therefore, elucidation of the biosynthesis of hopanoid side chains is in great interest for a better understanding of the physiological distribution and biological importance of bacteriohopanoids.In this work, ribosylhopane was isolated for the first time from a bacterium. This discovery is a solid proof for the role of ribosylhopane as an intermediate in the biosynthesis of hopanoid side chain. In addition, two genes involved in the hopanoid production in Streptomyces coelicolor have been characterized. Adnosylhopane may be converted into ribosylhopane by a phosphorylase; and an aminotransferase is required for the formation of aminobacteriohopantriol from ribosylhopane. Moreover, we have developed a concise strategy for the hemisynthesis of adenosylhopane and a deuteriated isotopmer. The subsequent incorporation of the deuteriated adenosylhopane into BHT by a cell-free system in…

In this work, the reactivity of acetylenic ω-ketoesters towards different metal complexes was investigated. When acetylenic ω-ketoesters are submitted to Ti(OiPr)4/iPrMgBr, the formation of fused bicyclic γ-hydroxy-α,β-unsaturated esters was observed. The products were obtained with absolute selectivity in regard to the ring junction formed (cis) and the configuration of the double bond (E) and could be transformed into the corresponding α,β-unsaturated lactones, substructures of various natural products. When ω-ketoalkynes are used in Ag(I)-catalyzed cycloisomerization reactions, the formation of spirocyclic compounds was observed. By taking advantage of the reaction intermediates of this reaction, it was possible to isolate the corresponding spirocyclic alkenyl iodides. Performing cycloisomerization reactions with acetylenic ω-ketoesters,…

This work describes the development of high-throughput droplet microfluidic platforms fine-tuned for protein of interest and their employment in directed evolution experiments. When not available, fluorogenic assay for monitoring desired enzyme activity (-ies) in droplets was developed. Moreover, the in vivo expression allowed the successive integration of microfluidic modules on the same chip. After a couple of evolution rounds the initial retro-aldolase variant was significantly improved. In other project, to meet industrial requirements a high-throughput screening platform for protease evolution in detergent has been assembled and validated. Two evolution rounds showed the accumulation of a certain pool of beneficial mutations over the selection rounds. The research described in this work highlighted that in vitro expression systems are sensitive to the amount of supplied DNA and reaction conditions. This observation led to the development of a multistep completely in vitro microfluidic platform.

One current formulation of Echinacea tablets which is examined in the present thesis is to produce tablets in a wet granulation process (WGP) with a high shear mixer. During the manufacturing, almost the whole amount of the excipient (lactose monohydrate) is wetted by Echinacea purpurea concentrate. In order to reduce the amount of excipients being granulated and dried by a basis granulate method was proposed where only a fraction of the total amount of filler (Microcrystalline cellulose, MCC) is used for granulation and drying, the rest of the filler (sorbitol) is added after granulation. This granulate can serve as basis material for…